The electrophoretic separation of protein mixtures has been a standard biochemical procedure for a generation of researchers. The most commonly used protocol, described by Laemmli, U. K. Nature, 227:680-685 (1970) takes advantage of observations made by Shapiro et al., Biochem, Biophys. Res. Commun., 28:815-820 (1967) and Weber and Osborn, J. Biol. Chem., 244:4406-4412 (1969) which showed that an anionic detergent, sodium dodecylsulfate (SDS), could be used for the separation of most proteins based on M.sub.r. Solubilization with SDS combined with a discontinuous polyacrylamide gel system, as originally described by Ornstein, Ann. NY Acad. Sci., 121:321-349 (1964) and Davis, Ann NY Acad. Sci., 121:404-427 (1964) allows the fine separation of dissociated proteins into discrete bands.
In the Laemmli system, SDS binding forms protein complexes that are nearly indistinguishable in an electric field. This is accomplished in two ways: SDS binds to most proteins in a constant ratio, 1.4 g of SDS per 1 g of protein, and imparts a constant charge to mass ratio to each protein so that their free mobilities are approximately equivalent; and SDS binding, in the presence of a reducing agent, causes a drastic structural change in the protein resulting in the formation of an SDS/protein complex shaped like a prolate ellipsoid with dimensions related to the M.sub.r, of the native protein. Since shape and charge density characteristics for all proteins in SDS are similar, separation based on M.sub.r is accomplished by electrophoresis through a support matrix with specific pore sizes The matrix acts as a sieve with smaller complexes moving through more easily than larger ones and, therefore, migrating further in the gel.
The characteristics of SDS/protein complexes (i.e., denatured conformation, constant charge to mass binding, and uniform shape) makes SDS the detergent of choice for many electrophoretic procedures, especially those involving the identification of subunits or in analyses of sample purity. Many researchers have come to rely on SDS-PAGE for the convenient assignment of M.sub.r based on relative mobilities; however, it is difficult to assess the biological activity of proteins treated with SDS, although several proteins have been shown to renature to an active form after detergent removal, Manrow and Dottin, Proc. Natl. Acad. Sci. USA, 77:730-734 (1980) and Scheele, Clin. Chem., 28:1056-1061 (1982). Another electrophoretic method, using the non-ionic detergent Tx-100, is commonly used in the production of zymograms, Hearing et al., Anal. Biochem, 72:113-117 (1976), unfortunately, this technique does not separate proteins based on size and the assignment of M.sub.r requires multiple runs at different gel concentrations, Tuan and Knowles, J. Biol. Chem., 259:2754-2763 (1984).
Akin et al. disclosed an entirely different electrophoretic system based on the cationic detergent CTAB that combines the most useful aspects of both the SDS and Tx-100 gels, Akin et al., Anal. Biochem., 145:170-176 (1985). Previous reports of electrophoresis systems, based on CTAB and related detergents, clearly demonstrate the separation of proteins as a logarithmic function of their M.sub.r, Eley et al., Anal. Biochem., 92:411-419 (1979), Panyim et al., Anal. Biochem., 81:320-327 (1977), Schick, Anal. Biochem., 63:345-349 (1975), and Marjanen and Ryrie, Biochem Biophys. Acta., 37:442-450 (1974). It has been shown that solubilization in CTAB does not cause loss of enzyme activity by denaturation, Spencer and Poole, J. Mol. Biol., 11:314-326 (1965). The work of Akin et al. further suggests that some proteins may be separated in a CTAB gel system and still retain native activity. For these gels, sample preparation is done without boiling and without the addition of reducing agent. Virtually all proteins prepared in this manner still migrate as a function of log M.sub.r ; however, in this continuous gel system resolution is less than optimal due to the absence of a stacking gel.